Name: transient gene expression in tobacco using gibson assembly and the gene gun
Uploaded: 2014-04-18T18:30:00
Description: Watch this Scientific Journal Video about Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun at JoVE.com

Forfatterne vil gerne takke Jen Sheen af Massachusetts General Hospital for den generøse donation af Nicotiana benthamiana frøplanter. Jen Bush hjulpet os meget i rådgivning i dyrkning af planter og oprettelse af en vækst kammer område. Tom Ferrante af Wyss Instituttet tilbød afgørende hjælp med konfokal mikroskopi. Forfatterne vil især gerne takke Don Ingber af Børnehospital Boston og Wyss Institut for den generøse donation af en Gene Gun og tilhørende forsyninger. Midler til projektet blev givet gennem en samarbejdsaftale med det amerikanske energiministerium Advanced Research Projects Agency (ARPA-E Award # DE-000.079) til PAS, JCW og MdM og gennem Chimerion Biotechnology, Inc. til MJV.

1.. Design af alternativt splejset sekvenser for Multiple Organelbiosyntese Targeting <ol><li> Bestem de steder til endelig protein udtryk. For dette arbejde, interessen i at målrette chloroplasten, peroxisom og cytosol. </li><li> Brug litteraturen til at identificere protein-og DNA-sekvenser, der vides at målrette proteiner organeller af interesse. Målretning I dette tilfælde en chloroplast sekvens fra Arabidopsis thaliana L-isoaspartate methyltransferase 6,15, og en peroxisom...

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgene+regulation+in+plants+by+alternative+splicing+of+a+suicide+exon.">Transgene regulation in plants by alternative splicing of a suicide exon.</a> Nucleic Acids Research. 40 (10), 4701-4710 (2012).</li><li>Syed, N. H., Kalyna, M., Marquez, Y., Barta, A., Brown, J. W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+in+plants+-+coming+of+age.">Alternative splicing in plants - coming of age.</a> Trends in Plant Science. 17 (10), 616-623 (2012).</li><li>Black, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+alternative+pre-messenger+RNA+splicing.">Mechanisms of alternative pre-messenger RNA splicing.</a> Annual Review of Biochemistry. 72, 291-336 (2003).</li><li>Dinkins, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+transcriptional+initiation+sites+and+alternative+5'-+and+3'-splice+site+selection+of+the+first+intron+deploys+Arabidopsis+protein+isoaspartyl+methyltransferase2+variants+to+different+subcellular+compartments.">Changing transcriptional initiation sites and alternative 5'- and 3'-splice site selection of the first intron deploys Arabidopsis protein isoaspartyl methyltransferase2 variants to different subcellular compartments.</a> The Plant Journal: for Cell and Molecular Biology. 55 (1), 1-13 (2008).</li><li>Kebeish, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplastic+photorespiratory+bypass+increases+photosynthesis+and+biomass+production+in+Arabidopsis+thaliana.">Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana.</a> Nature Biotechnology. 25 (5), 593-599 (2007).</li><li>Maier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+Introduction+of+a+Glycolate+Oxidative+Cycle+into+A.+thaliana+Chloroplasts+Leads+to+Growth+Improvement.">Transgenic Introduction of a Glycolate Oxidative Cycle into A. thaliana Chloroplasts Leads to Growth Improvement.</a> Frontiers in Plant Science. 3 (38), 1-12 (2012).</li><li>Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+the+isoprenoid+pathway+in+tobacco+by+expressing+the+cytoplasmic+mevalonate+pathway+in+chloroplasts.">Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts.</a> Metabolic Engineering. 14 (1), (2012).</li><li>Sapir-Mir, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+localization+of+Arabidopsis+isopentenyl+diphosphate+isomerases+suggests+that+part+of+the+plant+isoprenoid+mevalonic+acid+pathway+is+compartmentalized+to+peroxisomes.">Peroxisomal localization of Arabidopsis isopentenyl diphosphate isomerases suggests that part of the plant isoprenoid mevalonic acid pathway is compartmentalized to peroxisomes.</a> Plant Physiology. 148 (3), 1219-1228 (2008).</li><li>Voges, M. J., Silver, P. A., Way, J. C., Mattozzi, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+a+heterologous+protein+to+multiple+plant+organelles+via+rationally+designed+5'+mRNA+tags.">Targeting a heterologous protein to multiple plant organelles via rationally designed 5' mRNA tags.</a> Journal of Biological Engineering. 7 (20), (2013).</li><li>Gibson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+overlapping+DNA+fragments.">Enzymatic assembly of overlapping DNA fragments.</a> Methods in Enzymology. 498, 349-361 (2011).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6 (5), 12-16 (2009).</li><li>Gibson, D. G., Smith, H. O., Hutchison, C. A., Venter, J. C., Merryman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+of+the+mouse+mitochondrial+genome.">Chemical synthesis of the mouse mitochondrial genome.</a> Nature Methods. 7 (11), 901-903 (2010).</li><li>Lowenson, J. D., Clarke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+D-aspartyl+residues+in+polypeptides+by+the+erythrocyte+L-isoaspartyl/D-aspartyl+protein+methyltransferase.+Implications+for+the+repair+hypothesis.">Recognition of D-aspartyl residues in polypeptides by the erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.</a> The Journal of Biological Chemistry. 267 (9), 5985-5995 (1992).</li><li>Lanyon-Hogg, T., Warriner, S. L., Baker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+a+camel+through+the+eye+of+a+needle:+the+import+of+folded+proteins+by+peroxisomes.">Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes.</a> Biology of the Cell / under the auspices of the European Cell Biology Organization. 102 (4), 245-263 (2010).</li><li>Reumann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Arabidopsis+leaf+peroxisomes+reveals+novel+targeting+peptides,+metabolic+pathways,+and+defense+mechanisms.">Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms.</a> The Plant Cell. 19 (10), 3170-3193 (2007).</li><li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture.">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> J. Vis. Exp. (12), (2008).</li><li>Hollender, C. A., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+Fluorescence+Complementation+(BiFC)+Assay+for+Protein-Protein+Interaction+in+Onion+Cells+Using+the+Helios+Gene.">Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene.</a> (40), (2010).</li><li>Li, M. Z., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+homologous+recombination+in+vitro+to+generate+recombinant+DNA+via+SLIC.">Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC.</a> Nature Methods. 4 (3), 251-256 (2007).</li></ol>

I denne undersøgelse er simple strategier er beskrevet for at lokalisere et enkelt transgene proteiner til flere cellulære rum i planter. Målet var at designe konstruktion, som ville udtrykke et enkelt gen i mere end ét organel i Nicotiana benthamiana. Strategier omfatter rationelt design af GFP-baserede DNA-konstruktioner, Gibson samling, levering af plasmider bladceller med Gene Gun, og observation af resultaterne med konfokal mikroskopi. Tre forskellige kort, N-terminale tags...

Dette arbejde er en metabolisk engineering / syntetisk biologi projekt, hvori planteceller er manipuleret til at udtrykke en reporter protein i flere organeller men med kun en enkelt DNA-konstruktion. En fremgangsmåde til at målrette proteiner mere end ét sted involverer kloning flere genetiske kopier, der hver indeholder en anden lokalisering peptid. Hver kopi skal indføres ved successiv retransformation, eller alternativt ved tilbagekrydsning enkelt transformationer 1. Dette indebærer yderligere kloning, og er begrænset af en lokalisering tag per endestation. En anden måde til at lokalisere et protein til flere steder er gennem alternativ splejsning 2-5. RNA transkriberet fra et enkelt gen, men forskellige eksemplarer af transkriptet behandles forskelligt, ofte i mere end én måde per celle. Dette kan resultere i mere end en messenger-RNA i cellen transkriberet fra et enkelt gen. Disse forskellige Messengeboxenr RNA&#39;er kan kode for forskellige isoformer af det samme protein eller i tilfælde af en rammeskift et andet protein helt. Selv alternativ splejsning er blevet beskrevet i litteraturen i mange år, er virkningsmekanismer og bevares donor og receptor splejsningssitene kun bliver belyst for nylig 6. Da disse steder er ved at blive bedre beskrevet, de åbner muligheder for engineering. Plant metaboliske ingeniører står over for en udfordring, når der udtrykker et protein i flere organeller. For et protein, der er til at være lokaliseret på mere end en organel, skal teknikeren klone det samme gen flere gange, hver med en separat signalsekvens dirigere den til organel af interesse. For et enkelt gen i tre organeller, det er simpelthen tre gener. Men for en seks-gen stofskiftevej, dette udvider til 18 gener, en betydelig kloning indsats. Kombination af flere lokaliserings-sekvenser i en enkelt, alternativt splejset gen tegnificantly reducerer denne indsats. For eksempel, re-engineering photorespiration 7,8 og isoprenoid syntese 9,10 involverer både grønkorn og peroxisomer. I vores tilfælde tog vi fordel af splejsningssteder som observeret i en naturlig Arabidopsis thaliana, der er beskrevet tidligere 6. Vi rationelt redesignet mRNA-sekvensen forlader naturlige splejsningssteder alene, men placeret sekvenser, der ville kode chloroplast-eller peroxisom målretning tags inden for alternativt splejset introner (figur 1). Det udtrykte protein kan eller kan ikke have et mærke, afhængig af, om præ-mRNA, som kodede den blev udskåret som et intron (figur 1g og 1h). For mere information om udformningen af konstruktionerne, der præsenteres i dette arbejde, kan du se følgesvend artikel 11. Fordi dette er stadig en betydelig kloning indsats Gibson samling, en ny metode til at klone DNA-konstruktioner, er used i byggeriet. Gibson-metoden kan anvendes til enhver sekvens, uanset restriktionssites (figur 2) 12-14. Den specifikke blanding af enzymer giver mulighed for en et-trins, isotermisk forsamling. I denne fremgangsmåde er adskillige dobbeltstrengede lineære DNA-dele udformet således, at de har overlappende sekvenser af ~ 50 bp. Gibson samling enzym mix delvist fordøjer de lineære DNA-dele, udsætter enkeltstrenge af homologe sekvenser. Disse partielle enkeltstrengede sekvenser er re-udglødet i reaktionsblandingen, hvilket resulterer i en hurtig, et skridt, sekvens-uafhængig, ligering fri subkloning reaktion. Dette arbejde beskriver 1) rationel design alternativt splejsede konstruktioner til ekspression i planter grønkorn, peroxisomer og cytosol, 2) deres kloning ved hjælp af den nye ligatur-fri metode til Gibson forsamling, 3) deres levering i tobaksbladstrenge celler med Gene Gun, og 4) resultater, der viser organel målretning, som observeret med GFPog konfokal mikroskopi.

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oligonucleotide primers</td>
			<td style="width:191px;">IDT</td>
			<td style="width:119px;">(custom)</td>
			<td style="width:167px;">Design specifically for construct</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GeneBlocks</td>
			<td style="width:191px;">IDT</td>
			<td style="width:119px;">(custom)</td>
			<td>500 bp oligonucleotides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ApE software</td>
			<td style="width:191px;">U Utah</td>
			<td style="width:119px;">(download)</td>
			<td>http://biologylabs.utah.edu/jorgensen/wayned/ape/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MinElute kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">28004</td>
			<td>Used to purify PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAprep spin miniprep kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">27104</td>
			<td>Used to prepare cloning-appropriate amounts of plasmid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phusion Master Mix with GC Buffer</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">M0532S</td>
			<td>Used to PCR-amplify gene of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gibson assembly reaction mix</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">E2611L</td>
			<td>Master mix of the following 9 ingredients</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M Tris-HCl pH7.5</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:119px;">T1075</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M MgCl2</td>
			<td style="width:191px;">G Biosiences</td>
			<td style="width:119px;">82023-086</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTP mix</td>
			<td style="width:191px;">Fermentas</td>
			<td style="width:119px;">R0192</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M DTT</td>
			<td style="width:191px;">Fermentas</td>
			<td style="width:119px;">R0861</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PEG-8000</td>
			<td style="width:191px;">Affymetrix</td>
			<td style="width:119px;">19966</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NAD</td>
			<td style="width:191px;">Applichem</td>
			<td style="width:119px;">A1124,0005</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T5 exonuclease</td>
			<td style="width:191px;">Epicentre</td>
			<td style="width:119px;">T5E4111K</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phusion polymerase</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">F530S</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Taq DNA ligase</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">M0208L</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gibthon.org</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>Website to simplify calculations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plasmid PLUS Maxi kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">12963</td>
			<td>Used to prepare DNA for gene gun bullets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene Gun system</td>
			<td style="width:191px;">BioRad</td>
			<td style="width:119px;">165-2451</td>
			<td>Includes all parts necessary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nicotania benthamiana</td>
			<td style="width:191px;">(n/a)</td>
			<td style="width:119px;">(n/a)</td>
			<td>Gift of Jen Sheen, MGH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal microscope</td>
			<td style="width:191px;">Leica</td>
			<td style="width:119px;">SP5 X MP</td>
			<td>Imaging of resultant cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Deep well slides</td>
			<td style="width:191px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">71561-01</td>
			<td>Used for confocal imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">A Plasmid Editor (ApE) </td>
			<td style="width:191px;">University of Utah </td>
			<td style="width:119px;"></td>
			<td>http://biologylabs.utah.edu/jorgensen/wayned/ape</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gibthon:Ligation calculator</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>http://django.gibthon.org/tools/ligcalc/</td>
		</tr>
	</tbody>
</table>

transient gene expression in tobacco using gibson assembly and the gene gun

For at målrette en enkelt protein til flere subcellulære organeller, planter typisk duplikere de relevante gener, og udtrykke hvert gen separat med komplekse regulerende strategier, herunder differential initiativtagere og / eller signalsekvenser. Metaboliske ingeniører og syntetiske biologer interesserede i at målrette enzymerne til et bestemt organel står over for en udfordring: For et protein, der er til at være lokaliseret til mere end ét organel skal teknikeren klone det samme gen flere gange. Dette arbejde viser en løsning på denne strategi: udnytte alternativ splejsning af mRNA. Denne teknologi udnytter etablerede kloroplast og peroxisome targetingsekvenser og kombinerer dem til et enkelt mRNA, der er alternativt splejsede. Nogle splejsningsvarianter sendes til chloroplasten, nogle til peroxisom, og nogle til cytosolen. Her er designet til flere organel målrettet med alternativ splejsning. I dette arbejde blev GFP forventes at være udtrykdede i chloroplasten, cytosol og peroxisom af en række rationelt konstrueret 5 &#39;mRNA tags. Disse tags har potentiale til at reducere mængden af ​​kloning kræves, når heterologe gener skal udtrykkes i flere subcellulære organeller. Konstruktionerne blev udformet i tidligere arbejde 11, og blev klonet under anvendelse Gibson samling, en ligering uafhængig kloning metode, som ikke kræver restriktionsenzymer. De resulterende plasmider blev indført i Nicotiana benthamiana epidermal bladceller med en modificeret Gene Gun protokol. Endelig blev transformerede blade observeret med konfokal mikroskopi.

Designet indsats var et resultat af en betydelig planlægning. Novel til dette projekt er brugen af ​​alternativ splejsning at skabe en præ-mRNA, der er oversat til differentielt udtrykte proteiner. Disse proteiner udtrykkes i forskellige organeller, i dette tilfælde chloroplasten, peroxisom og / eller cytosol. Vi tilpasset naturlige Arabidopsis gen, der alternativt splejsede 6 og placerede kendt kloroplast 6 og peroxisom 17 targetingsekvenser i alternative exoner (TriTag-...

Watch this Scientific Journal Video about Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun at JoVE.com

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Dette arbejde beskriver en hidtil ukendt fremgangsmåde til selektivt at målrette subcellulære organeller i planter, analyseret under anvendelse af BioRad Gene Gun.

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Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

Transcript og Metabolit Profilering for Vurdering af tobak Tree og Poppel som råvare til Bio-industri

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Sporing mikrobiel forurening i Retail Miljøer med fluorescerende Powder - En Retail Delicatessen Environment Eksempel

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Ekspression af rekombinant Cellulase Cel5A fra<em&gt; Trichoderma reesei</em&gt; I tobaksplanter

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Screening Levnedsmidler for klasse 1 Integrons og genkassetter

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

RNAi-medieret Kontrol af aflatoksiner i Peanut: metode til at analysere Mykotoksin Produktion og transgenekspression i Peanut /<em&gt; Aspergillus</em&gt; Pathosystem

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacle Balanus improvisus som en Marine Model - dyrkning og genekspression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium tumefaciens og Agrobacterium rhizogenes-medieret Transformation af kartoffel og promotor aktivitet af en Suberin gen af GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

Aftagne blad analyser for at forenkle genekspression undersøgelser i kartoffel under infestation ved at tygge insekt Manduca Sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Beregning af bladareal ved hjælp af tre forskellige metoder i rene løvfældende stande

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...